The human lens must maintain transparency over many decades and, to do so, lens fiber cells must minimize extracellular space and preserve protein solubility. Lens membrane proteins, such as channels and adhesion molecules, play essential roles in lens development and maintenance of lens transparency. As fiber cells mature and age, lens proteins undergo post- translational modification that may alter protein function to maintain lens homeostasis or may lead to cataracts. Aquaporin-0 (AQP0) is the most abundant lens membrane protein with reported roles in fiber cell adhesion, in water permeability, and in fiber cell organization and, as such, plays important roles in the development and maintenance of lens transparency. The long-term goals of our research are to identify modifications to the lens membrane proteome during development, aging, and cataractogenesis and to understand how lens membrane protein function is altered by modification. Our general hypothesis is that modifications to lens membrane proteins alter protein function in specific lens regions during lens development and accumulate with age leading to cataracts. Specifically, we hypothesize that the multiple functions of AQP0 are controlled by specific posttranslational modifications that alter membrane targeting, permeability, and cytoskeletal interaction. In addition, we hypothesize that a second lens water channel, AQP5, serves as a rescue channel in response to osmotic stress. To test our hypotheses we will employ proteomics and imaging approaches to obtain a molecular level understanding of lens membrane proteome changes during fiber cell development and aging. We will then use functional assays to determine how modifications affect membrane protein function. We propose three aims: 1) to spatially map the age- and cataract-related changes to the human lens membrane proteome, 2) to determine the role of AQP0 in lens cell morphology and membrane organization during fiber cell differentiation and aging, and 3) to determine the role of AQP0 and AQP5 in fiber cell water permeability in specific lens regions and in response to osmotic stress. The global approach proposed is expected to provide new molecular level information on the structure and function of the most abundant lens membrane proteins. PUBLIC HEALTH RELEVANCE: Narrative The relevance of the proposed studies is that with an improved understanding of normal lens development, aging processes, and cataractogenesis, therapies can be developed to better treat or delay the onset of cataract in our increasingly aging population;thereby extending clear vision in this population. In addition, the expected long-term outcomes will have a significant impact on the enormous financial costs of cataract treatment.